CBFβ-SMMHC, expressed in M4Eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells

Abstract

CBFb-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBFb-SMMHC or AML1-ETO oncoproteins failed to develop de®nitive hematopoiesis. To investigate these e ects on hematopoiesis, we expressed CBFb-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBFb-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBFb. Indirect immuno¯uorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and di use nuclear staining in 32D cl3 cells. CBFb-SMMHC reduced endogenous CBF DNA-binding ®vefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBFb-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBFb-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor a or b subunit levels. Neither apoptosis nor 32D di erentiation was induced by zinc in IL-3 in these lines. Induction of CBFb-SMMHC in 32D cl3 cells did not inhibit their di erentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of di erentiation by CBFb-SMMHC by allowing its increased expression.

Identification of a region on the outer surface of the CBFβ-SMMHC myeloid oncoprotein assembly competence domain critical for multimerization

“…Stable transfectants were selected by limiting dilution in 96-well dishes in the presence of 1.2 mg/ml G418 (total). Zinc chloride was employed at 100 mM, and cell cycle analysis was carried out as described by pulsing for 30 min with bromodeoxyuracil (BrdU) followed by fixation and fluorescein isothiocyanateanti-BrdU and propidium iodide staining as described (Cao et al, 1997).…”

“…Cytoplasmic proteins were concentrated by precipitation using four volumes of acetone at À201C. Nuclear and cytoplasmic extract corresponding to equivalent numbers of cells were subjected to Western blotting using rabbit anti-CBFb antiserum as described (Cao et al, 1997). Band intensities were quantified using NIH ImageJ 1.33 software.…”

“…CBFb-SMMHC inhibits G1 to S cell cycle progression of hematopoietic cell lines or murine myeloid progenitors dependent upon residues in the CBFb domain that contact AML1 (Cao et al, 1997(Cao et al, , 1998D'Costa et al, 2005). AML1-ETO also slows proliferation (Burel et al, 2001), and exogenous AML1 accelerates G1 dependent upon its transactivation domain (Strom et al, 2000;Bernardin and Friedman, 2002).…”

The chimeric protein, PEBP2β/CBFβ-SMMHC, disorganizes cytoplasmic stress fibers and inhibits transcriptional activation

“…A carboxy-terminal portion of the chimera contains the rod domain of the myosin heavy chain molecule which is responsible for the molecule's oligomerization (Kiehart, 1990). An aminoterminal portion of the chimera harbors most of the amino acid sequence of the PEBP2b/CBFb protein, and the chimeric polypeptide itself has been shown to retain the ability to interact with the a subunit of PEBP2/CBF (Cao et al, 1997;Liu et al, 1994Liu et al, , 1996. It is also reported that the chimeric protein can deregulate PEBP2/CBF site dependent transcriptional activation in transient expression assays .…”

“…As for the PEBP2b/CBFb-SMMHC chimeric protein, its subcellular localization appears to be either cytoplasmic and/or nuclear, depending on the experimental conditions employed in each report (Cao et al, 1997;Liu et al, 1996;Lu et al, 1995;Wijmenga et al, 1996). In order to extend our previous observations on the cytoskeleton-a nity of the PEBP2b/CBFb protein , we have focused one part of the present study on the possible e ects of the cytoplasmic PEBP2b/CBFb-SMMHC protein on cell morphology and F-actin containing stress ®bers.…”